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, 218 (1), 192-8

Blockade of sphingosine-1-phosphate Reduces Macrophage Influx and Retinal and Choroidal Neovascularization

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Blockade of sphingosine-1-phosphate Reduces Macrophage Influx and Retinal and Choroidal Neovascularization

Bing Xie et al. J Cell Physiol.

Abstract

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule that stimulates endothelial cell migration, proliferation, and survival in vitro, and tumor angiogenesis in vivo. In this study, we used a humanized monoclonal antibody (sonepcizumab) that selectively binds S1P to investigate its role in retinal and choroidal neovascularization (NV). Intraocular injection of sonepcizumab significantly reduced macrophage influx into ischemic retina and strongly suppressed retinal NV in mice with oxygen-induced ischemic retinopathy. In mice with laser-induced rupture sites in Bruch's membrane, intraocular injection of sonepcizumab significantly reduced the area of choroidal NV and concomitantly reduced fluorescein leakage from the remaining choroidal NV. Four weeks after intraocular injection of up to 1.8 mg of the sonepcizumab in non-human primates, electroretinograms and fluorescein angiograms were normal, and light microscopy of ocular sections showed no evidence of structural damage. These data show for the first time that S1P stimulates both choroidal and retinal NV and suggest that sonepcizumab could be considered for evaluation in patients with choroidal or retinal NV.

Figures

Figure 1
Figure 1. Measurement of radioactivity in ocular tissues 1, 7 and 14 days after Intravitreous injection of [3H]sonepcizumab
Four week old C57BL/6 mice were given an intravitreous injection of [3H] sonepcizumab in each eye and euthanized 1, 7, or 14 days after the injection. Eyes were removed and lens, retina, and eyecup consisting of the retinal pigmented epithelium, choroid, and sclera were dissected and homogenized. The protein concentration was measured and radioactivity was counted in a scintillation counter. Each bar represents the mean (±SEM) calculated from 20 eyes and values are represented as [3H] CPM/mg tissue. Lens, retinas and eyecups dissected from the mice exhibited the highest level of total radioactivity per milligram of tissue at day 7 after intravitreous dosing of [3H]sonepcizumab. The level of radioactivity was significantly higher at day 7 than at day 1 and day 14. Levels in retina and eyecup were comparable. Statistical comparisons were made by ANOVA with Dunnett’s correction for multiple comparisons, (P<0.05).
Figure 2
Figure 2. Intravitreous sonepcizumab suppresses choroidal neovascularization (NV)
Five - six week old C57BL/6 mice were given an intravitreous injection in each eye consisting of 1 μl containing 0.05, 0.5, 1.0, or 3.0 μg of sonepcizumab or PBS 1 day before rupture of Bruch’s membrane at 3 locations with laser photocoagulation. After two weeks, the mice were perfused with fluorescein-labeled dextran and choroidal flat mounts were examined by fluorescence microscopy. Compared to eyes injected with PBS (A), those injected with 0.05 μg of LT1009 (B) showed similar appearing choroidal NV at Bruch’s membrane rupture sites, but eyes injected with 0.5 (C), 1.0 (D), or 3.0 (g (E) seemed to show smaller areas of choroidal NV. The area of choroidal NV at each rupture site was measured by image analysis and the mean area of choroidal NV per eye was calculated to give a single experimental value. In (F), the bars show the mean (±SD) for each group calculated from 20 experimental values and confirm that compared to eyes injected with PBS, the area of choroidal NV was significantly less in eyes injected with 0.5, 1.0, or 3.0 (μg of LT10029 (which were not significantly different from each other), but not those injected with 0.05 (μg of LT10029. *p = 0.01 for difference from PBS by ANOVA with Dunnett’s correction for multiple comparisons.
Figure 3
Figure 3. LT1009 reduces leakage from choroidal neovascularization (NV) at Bruch’s membrane rupture sites
C57BL/6 mice (n = 10) had rupture of Bruch’s membrane in 3 locations in each eye were given an intraocular injection of 3 μg of sonepcizumab in one eye and PBS in the fellow eye. After one week, the mice were given an intraperitoneal injection of 12 μl/g body weight of 1% fluorescein sodium and 5 minutes later, retinas were dissected, stained with anti-PECAM-1, and retinal flat mounts were examined by fluorescence microscopy. The area of fluorescein and PECAM-1 staining was measured by image analysis by a masked investigator and the area of leakage was calculated by subtracting the latter from the former. Eyes injected with sonepcizumab showed smaller areas of fluorescein staining (A) compared to eyes injected with PBS (B). They also showed smaller areas of PECAM-1 staining (C versus D). Merged images from sonepcizumab-injected (E) and PBS-injected (F) eyes illustrate double labeling (yellow) in areas of choroidal NV and fluorescein that has leaked into surrounding tissue (F). Compared to eyes injected with PBS, those injected with sonepcizumab showed significantly smaller areas of PECAM-1 staining at Bruch’s membrane rupture sites (G) and significantly less leakage (H). Statistical comparisons were made by unpaired t-test..
Figure 4
Figure 4. Intravitreous sonepcizumab suppresses ischemia-induced retinal neovascularization (NV)
C57BL/6 mice were placed in 75% oxygen at postnatal day (P) 7 and at P12 they were returned to room air and given an intravitreous injection of 3 μg of sonepcizumab in one eye and PBS in the fellow eye. At P17, mice were given an intravitreous injection of rat anti-mouse PECAM-1 antibody and euthanized after 12 hours. Retinas were removed, washed, and incubated in 1:500 FITC-labeled goat anti-rat IgG and retinal flat mounts were examined by fluorescence microscopy. Retinas from mice treated with sonepcizumab (A) appeared to have less NV than retinas from mice treated with PBS (B). Image analysis by a masked investigator confirmed that compared to fellow eyes injected with PBS, eyes injected with sonepcizumab (n=7 for each) showed a significant reduction in mean (±SD) area of NV per retina (C). Statistical comparison was made by unpaired t-test.
Figure 5
Figure 5. Intravitreous injection of sonepcizumab reduces influx of macrophages into ischemic retina
C57BL/6 mice (n = 8) were placed in 75% oxygen at postnatal day (P) 7 and at P12 they were returned to room air and given an intraocular injection of 3 μg sonepcizumab in one eye and PBS in the fellow eye. At P17, mice were given an intravitreous injection of F4/80 antibody labeled with Alexa and after 8 hours retinal flat mounts were prepared and examined by fluorescence microscopy. Compared to fellow eyes treated with PBS which showed dense infiltration with macrophages (A), there were fewer macrophages in retinas from eyes injected with sonepcizumab (B). This is most easily seen in the insets which are magnified views of the boxed areas. Image analysis confirmed that there was a significant reduction in the mean (±SD) number of macrophages per retina in eyes treated with sonepcizumab compared to those treated with PBS (C, n = 8 for each). Statistical comparisons were made by unpaired t-test.

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